1. Field of the Invention
The present invention relates to a method for determining the concentration of particles dispersed and moving at random in a sample solution, by using an optical system which can detect light from a micro region in a solution such as an optical system of a confocal microscope or a multiphoton microscope.
Priority is claimed on Japanese Patent Application No. 2011-092168, filed Apr. 18, 2011, the content of which is incorporated herein by reference.
The present application is a U.S. continuation application based on the PCT International Patent Application, PCT/JP2012/060487, filed on Apr. 18, 2012, the contents of which are incorporated herein by reference.
2. Description of the Related Art
According to the developments in photometric measurement techniques in recent years, detection/measurement of faint light at a single photon or single fluorescent molecule level have become possible by using an optical system of a confocal microscope and a super high sensitive light detection technique capable of the photon counting (single photon detection). Thus, various devices or methods of performing the detection of intermolecular interaction or binding/dissociating reaction of biological molecules, etc. by using such a faint light measurement technique, have been proposed. For example, in Fluorescence Correlation Spectroscopy (FCS, see e.g. Japanese Unexamined Patent Application, First Publication No. 2005-098876; Japanese Unexamined Patent Application, First Publication No. 2008-292371; Masataka Kinjo; “Protein, Nucleic acid, and Enzyme” Vol. 44, No. 9, pages 1431-1438, 1999.; Meyer-Alms; “Fluorescence Correlation Spectroscopy” edt. R. Rigler, Springer, Berlin, pages 204-224, 2000.; and Noriko Kato, et al. “Gene & Medicine”, Vol. 6, No. 2, pages 271-277, 2002.), by using an optical system of a laser confocal microscope and a photon counting technique, the measurement is performed on the fluorescence intensity from fluorescence molecules or fluorescently labeled molecules (fluorescent molecules, etc.), entering and exiting a micro region in a sample solution (the focal region to which the laser light of the microscope is condensed, called a “confocal volume”). Based on the average dwell time (translational diffusion time) of the fluorescent molecules, etc. and the average number of the dwelling molecules in the micro region, determined from the autocorrelation function value of the measured fluorescence intensity, the information, such as the speed of the movement, the size, and the concentration of the fluorescent molecules, etc. are acquired, or various phenomena, such as a change of a molecular structure or size, a binding/dissociating reaction, and dispersion/aggregation of molecules, are detected. Moreover, in Fluorescence Intensity Distribution Analysis (FIDA, e.g. Japanese Patent No. 4023523) or Photon Counting Histogram (PCH, e.g. PCT International Publication No. WO 2008-080417), a histogram is made on the fluorescence intensity of fluorescent molecules, etc., entering and exiting a confocal volume, measured similarly to FCS. The average value of the characteristic brightness of the fluorescent molecules, etc. and the average number of molecules dwelling in the confocal volume are calculated by fitting a statistical model formula to the distribution of the histogram. Based on the information thereof, the change of the molecular structure or size, binding/dissociative conditions, and dispersion/aggregation conditions of molecules are estimated. Furthermore, Japanese Unexamined Patent Application, First Publication No. 2007-20565 and Japanese Unexamined Patent Application, First Publication No. 2008-116440 propose methods of detecting fluorescent substances based on a time progress of a fluorescence signal of a sample solution measured by using an optical system of a confocal microscope. In Japanese Unexamined Patent Application, First Publication No. H04-337446, faint light from fluorescent fine particles flowing through a flow cytometer or fluorescent fine particles fixed on a substrate is measured by using a photon counting technique. Moreover, there is proposed a signal calculation processing technique for detecting the existences of the fluorescent fine particles in the flow or on the substrate from the faint light.
Especially, according to the method employing the fluorescence measurement technique of a micro region by using an optical system of a confocal microscope and a photon counting technique, such as FCS and FIDA, the concentration and the amount of the sample required for the measurement may be extremely small (an amount used in one measurement is about several tens of μL at most), as compared with the prior art, and the measuring time is also shortened a lot (in one measurement, a measuring process taking a time of a second order is repeated several times). Thus, those techniques are expected to be a strong tool enabling an experiment or a test at low cost or quickly in comparison with conventional biochemical methods, especially in conducting an analysis of rare or expensive samples which are often used in the field of medical or biological research and development, or in conducting a test of a large number of specimens, such as clinical diagnosis of diseases or the screening of bioactive substances.